PROJECT SUMMARY To contribute to the defeat of tuberculosis, we focus on understanding three families of small protein complexes, which are dramatically expanded within pathogenic Mycobacterial species and are believed to be among the most critical for bacterial survival and pathogenesis. We propose X-ray structural studies of complexes because these reveal how protein machines work, and how they may be disrupted. Our targets are PE/PPE complexes, associated with the cell wall and the host immune response; Esx complexes, associated with type VII secretion systems; and toxin-antitoxin complexes of which Mtb contains 88 pairs. We have developed and tested innovative methods: a computational method (PROLINKS) for predicting which pairs of proteins participate in a complex; a robust experimental method for validating predicted complexes for crystallographic studies; and new methods for optimizing crystallization (SER server). These methods allow us to determine structures of protein complexes, rather than individual molecules. We have achieved proof of principle structures for all three of our targets , and have clones and expression systems ready to rapidly expand knowledge of these potentially vulnerable Mtb complexes. In addition, we propose structural studies of the potentially vulnerable polyketide synthase system, specifically Pks13, for which we will seek structures of enzyme-substrate complexes. Through collaborative work with the Genetics and Chemical Core, the TAMU project, and our collaborators. Dr. Robert Modlin of UCLA and Dr. William Jacobs of Einstein, we will seek functional information on the complexes that we prepare. We will continue to collaborate with the Structure Determination Core on the development of methods, and will call on their facility when our structural problems exceed local capacity.